Abstract
Besides the acquisition of pharmacokinetic parameters of antisense oligonucleotide microRNA (miRNA) inhibitors, such as measuring in vivo concentration, their pharmacodynamic characteristics are also of interest. An emerging and straightforward method for studying molecular interactions is microscale thermophoresis (MST). This technique makes it possible to study interactions between miRNAs and various oligonucleotide inhibitors, independent of the chemical modifications of the inhibitors or their respective target structure, with very little sample volume required compared to competitive techniques, such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Interaction studies between these inhibitors and their respective target structures were performed, and they allowed the assessment of binding characteristics and parameters, such as EC50 for a number of these inhibitors, with little effort. Furthermore, MST could be utilized for obtaining kinetic binding data of the Argonaute-2 protein with a miRNA, which showed a possible RNA-induced silencing complex (RISC)-mediated turnover of inhibited miRNAs.
Highlights
Noncoding RNAs have recently been identified as powerful targets to combat many different diseases.[1]
An anti-miRNA132 library was designed, which besides sequence homology contained different chemical modifications. 2’-O-Methyl modification (OMe) was chosen as ribose modification to study its influence on binding affinity (Table 1)
The capillary scan, capillary shape, microscale thermophoresis (MST) traces, and the binding curve of OMe132_S5 with miRNA132 are shown as examples (Figure 2), while the remaining binding curves can be found in the Supplemental Information
Summary
Noncoding RNAs have recently been identified as powerful targets to combat many different diseases.[1]. A large variety of techniques are available, such as X-ray crystallography, nuclear magnetic resonance (NMR), mass spectrometry (MS), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and thermal shift assays.[3,4] Those methods vary in terms of high-throughput ability, complexity, and the amount of sample needed. Those assays are very robust and reproducible, and they possess a low sample consumption. One emerging method to assess pharmacological parameters, such as the dissociation constant (KD), is microscale thermophoresis (MST). The basic mode of action of an MST experiment is illustrated below (Figure 1)
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